Several recent studies highlight the possibility of using multipotent adult bone marrow-derived mesenchymal stem cells (MSCs) for tissue engineering applications, and certain soluble factors (such as EGF) have been identified and characterized for their ability to promote MSC viability and differentiation into specific tissue types;however, much less is known about the role of the scaffold, designed to mimic the native ECM, in regulating the behavior of these stem cells. It is becoming increasingly clear that the ECM contains several factors, such as mechanical integrity, adhesion specificity, and growth factor availability, which are all individually and collectively critical in dictating the local cell and tissue behavior. The hypothesis of this proposal is that by independently tuning the stiffness, ligand identity and EGF-availability of the ECM, MSC behavior and signaling can be tightly regulated. Similar changes in the physical state of the microenvironment on 2-D surfaces have been previously shown to alter MSC phenotype. However, the signaling mechanism involved in these phenotypic changes remains unclear, as does how these cells will react to these factors in a 3-D environment. In addition, previous studies by the sponsors'laboratories have elucidated the importance of the EGF receptor in the enhancement of MSC viability on 2-D surfaces. This proposal aims to test this hypothesis in a novel 3-D environment that contains the versatility to independently control all of these parameters. To do this, this proposal includes the design of 3-D hydrogel constructs based on polyethylene glycol with tunable mechanical properties, the ability to tailor the specific ECM protein of interest to facilitate cell adhesion, and simple methods to control the availability of EGF. MSCs will be seeded throughout the bulk of these synthetic matrices and cultured for defined periods of time relevant for cellular responses specific to MSC behavior. During these studies, MSC apoptosis, proliferation, and lineage specification will be examined as a function of ECM crosslinking and ligand identity, as well as how this switch is affected by modulating EGFR-mediated signaling. The biophysical design criteria resulting from these studies will potentially have a far-reaching impact on the field of tissue engineering and in the future design of smart biomaterials to direct cell behavior.